Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) Honey

Guang Yi Wang, Themis J. Michailides, Bruce D. Hammock, Young Moo Lee, Richard M. Bostock

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone (MBTFP), was found to competitively inhibit cutinase activity (I50 = 9.4 x 10-3) from the fungal plant pathogen Monilinia fructicola and to serve as an effective affinity ligand for the purification of cutinases from culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1-trifluoro-2-propanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I50 values of 1.6 x 10-6 and 2.3 x 10-4 M, respectively. Buffer containing OTFP was the strongest eluant for cutinases of M. fructicola and provided the best purification factor and yield, although buffers containing OTFP, detergent, and salt were found to be effective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity column. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric focusing of the affinity-purified cutinase fraction indicated activity associated with proteins of pI 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [3H]cutin and artificial substrates such as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragments were separated by Microbore-HPLC. The amino acid sequences of several internal peptide fragments had high homology with cutinase sequences from other fungi, particularly the plant pathogen Botrytis cinerea. Our study illustrates the potential of TFK ligands for the affinity purification of cutinases and indicates that the cutinases from M. fructicola have novel features warranting further study. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)31-38
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume382
Issue number1
DOIs
StatePublished - Oct 1 2000

Fingerprint

Honey
Pathogens
Purification
Acetone
Ketones
Buffers
Peptide Fragments
Molecular mass
cutinase
Gels
Ligands
Botrytis
Proteins
Electrophoresis, Gel, Two-Dimensional
Octoxynol
Isoelectric Focusing
Hydrolases
Electrophoresis
Fungi
Detergents

Keywords

  • Affinity purification
  • Cutinase
  • Monilinia fructicola
  • Serine hydrolases
  • Trifluoromethyl ketone

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) Honey. / Wang, Guang Yi; Michailides, Themis J.; Hammock, Bruce D.; Lee, Young Moo; Bostock, Richard M.

In: Archives of Biochemistry and Biophysics, Vol. 382, No. 1, 01.10.2000, p. 31-38.

Research output: Contribution to journalArticle

Wang, Guang Yi ; Michailides, Themis J. ; Hammock, Bruce D. ; Lee, Young Moo ; Bostock, Richard M. / Affinity purification and characterization of a cutinase from the fungal plant pathogen Monilinia fructicola (Wint.) Honey. In: Archives of Biochemistry and Biophysics. 2000 ; Vol. 382, No. 1. pp. 31-38.
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AU - Bostock, Richard M.

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AB - Trifluoromethyl ketones (TFK) are potent inhibitors of a variety of serine hydrolases. The TFK inhibitor, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone (MBTFP), was found to competitively inhibit cutinase activity (I50 = 9.4 x 10-3) from the fungal plant pathogen Monilinia fructicola and to serve as an effective affinity ligand for the purification of cutinases from culture filtrates. The TFK inhibitors, 3-n-octylthio-1,1,1-trifluoro-2-propanone (OTFP) and 3-n-pentylthio-1,1,1-trifluoro-2-propanone (PTFP), also inhibited cutinase activity with I50 values of 1.6 x 10-6 and 2.3 x 10-4 M, respectively. Buffer containing OTFP was the strongest eluant for cutinases of M. fructicola and provided the best purification factor and yield, although buffers containing OTFP, detergent, and salt were found to be effective for eluting cutinases bound to MBTFP-Sepharose. Buffer containing 0.5% Triton X-100 also selectively eluted cutinases from the affinity column. Two-dimensional electrophoretic analysis by SDS-PAGE and isoelectric focusing of the affinity-purified cutinase fraction indicated activity associated with proteins of pI 8.2 and molecular masses of approximately 18.6 and 20.8 kDa. These proteins hydrolyzed [3H]cutin and artificial substrates such as p-nitrophenylbutyrate and related esters, typical of other cutinases, but differ from previously characterized cutinases in molecular mass. The two low-molecular-weight proteins resolved by 2-D gel electrophoresis were subjected to in-gel digestion with Lys-C and the resulting peptide fragments were separated by Microbore-HPLC. The amino acid sequences of several internal peptide fragments had high homology with cutinase sequences from other fungi, particularly the plant pathogen Botrytis cinerea. Our study illustrates the potential of TFK ligands for the affinity purification of cutinases and indicates that the cutinases from M. fructicola have novel features warranting further study. (C) 2000 Academic Press.

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